A new therapeutic modality is to use oligonucleotides to restore the levels of tumor suppressive miRNAs to that of normal cells or tissues. Successful targeting and delivery of therapeutic oligonucleotides has been a major bottleneck in their clinical development. Microvesicles are a small homogenous subtype of membrane vesicles of endocytic origin and naturally contain a variety of cellular biochemicals including miRNA. This proposal will develop an microvesicle drug delivery system to target therapeutic miRNAs to hepatocellular carcinoma cells and tumors. A unique feature of this system is that the nucleic acid cargo is synthesized by the cells that produce the microvesicles, thus alleviating the need for synthetic oligonucleotides. Microvesicles will be engineered to express a targeting peptide that will direct it to cancer cells. The modified pre-miR-199a, engineered to include the loop region of the TAR RNA hairpin, will be inserted into an intron of the targeting protein gene and once spliced and processed, will be directed to the microvesicle by binding to C-terminus HIV Tat peptide on the targeting protein. This project will use in silico and biochemical approaches to determine the optimal pre-miRNA sequences for peptide binding, correct processing and biological activity. The therapeutic activity and targeting ability of the microvesicle delivery system will be evaluated in vitro and in an orthotopic model of hepatocellular carcinoma. Pharmacodynamic and pharmacokinetic evaluations will provide detailed knowledge on dose, toxicity, efficacy, route of administration and biodistribution. Proposed here is a highly innovatie approach to synthesize and deliver therapeutic nucleic acids to cancer cells. This system does not rely upon the use of synthetic oligonucleotides or artificial drug delivery systems. Future manifestations of this technology can be applied to deliver other nucleic acid drugs such as shRNA and may be widely applicable to treat many diseases.